Cell-type and sex-specific rhythmic gene expression in the nucleus accumbens

Abstract

Circadian rhythms are critical for human health and are highly conserved across species. Disruptions in these rhythms contribute to many diseases, including psychiatric disorders. Previous results suggest that circadian genes modulate behavior through specific cell types in the nucleus accumbens (NAc), particularly dopamine D1-expressing medium spiny neurons (MSNs). However, diurnal rhythms in transcript expression have not been investigated in NAc MSNs. In this study we identified and characterized rhythmic transcripts in D1- and D2-expressing neurons and compared rhythmicity results to homogenate as well as astrocyte samples taken from the NAc of male and female mice. We find that all cell types have transcripts with diurnal rhythms and that top rhythmic transcripts are largely core clock genes, which peak at approximately the same time of day in each cell type and sex. While clock-controlled rhythmic transcripts are enriched for protein regulation pathways across cell type, cell signaling and signal transduction related processes are most commonly enriched in MSNs. In contrast to core clock genes, these clock-controlled rhythmic transcripts tend to reach their peak in expression about 2-h later in females than males, suggesting diurnal rhythms in reward may be delayed in females. We also find sex differences in pathway enrichment for rhythmic transcripts peaking at different times of day. Protein folding and immune responses are enriched in transcripts that peak in the dark phase, while metabolic processes are primarily enriched in transcripts that peak in the light phase. Importantly, we also find that several classic markers used to categorize MSNs are rhythmic in the NAc. This is critical since the use of rhythmic markers could lead to over- or under-enrichment of targeted cell types depending on the time at which they are sampled. This study greatly expands our knowledge of how individual cell types contribute to rhythms in the NAc.

Figure 1.. Rhythmicity in the NAc varies by cell type.

(A) Table listing numbers of rhythmic transcripts identified in each cell type in the NAc at various thresholds of significance (p values and q values (Benjamini-Hochberg correction). (B) Scatterplots show expression patterns (logCPM) across time of day for top rhythmic transcripts. These six core clock genes have consistent peak times across cell type. (C) For each cell type, pathway analyses identified pathways of rhythmic transcripts, which reached a significance threshold of p<0.05. The top 5 enriched pathways from each cell type are plotted together.

Figure 2.. Rhythmic transcripts vary by across cell type and sex in the NAc.

(A) Table listing numbers of rhythmic transcripts identified at various significance thresholds in each cell type for male and female samples taken from the NAc. (B) Venn diagrams are used to show overlap across sex for rhythmic transcripts at multiple significance thresholds (p<0.05 (left) or p<0.10 (right)) in D1 and D2 cells. (C) RRHO plots are also used to visualize overlap across sex in a threshold-free manner. (D) Heatmaps of rhythmic transcripts show patterns in peak times across groups. (E-F) Pathway analyses identified significant (p<0.05) pathways enriched for rhythmic transcripts. The top 5 enriched pathways from each cell type are plotted together for (E) female and (F) male samples separately.

Figure 3.. Rhythmic transcripts tend to peak later in females than males.

(A) Peak times for each rhythmic transcript are shown in radar plots for each cell type in males and females across the 24-hour clock (ZT). The time with the highest proportion of peaking genes, as well as its opposite (12-hours apart), is indicated with a dot. Separate radar plots are shown for rhythmic transcripts that are (B) overlapping or (C) unique in males and females. (D) For genes that overlap in males and females, we also show phase concordance plots and (E) quantified the difference in peak time. On average, transcripts peaked 2 hours later in D1 and D2 cells taken from females compared to males. (F) Example transcripts with varying peak times across sex are shown with smoothed curves as well as individual data points: (left) ~6 hour, (middle) ~12 hour and (right) ~2-hour difference.

Figure 4.. Rhythmic transcripts peaking in the light phase contribute to largely distinct enriched pathways.

(A-D) Pathway analyses identified significant pathways (p<0.05) enriched for rhythmic transcripts falling into clustered peaks (“light” and “dark”). The tope 5 enriched pathways from each cell type are plotted together for each sex and peak time. For proper scale, p-values are truncated at -log10(p-value)≤5 even if the pathway is more significantly enriched.

Figure 5.. Rhythmicity should be considered when cell-type specific markers are selected.

(A) Table showing rhythmicity (p-values and peak times) for several traditional markers used to distinguish D1- and D2-expressing neurons. (B) To visualize these rhythms, rhythmic markers from Table A are shown as smoothed curves in expression (logCPM) and individual data points in both D1 cells (rhythmic) and D2 cells (not significantly rhythmic). (C) Novel transcripts that have been identified for cell-type differentiation in the NAc are also found in our analysis. A subset of these are rhythmic, with many peaking in the dark phase. (D) The top 5 differentially expressed, non-rhythmic transcripts (rhythmicity p-value<0.10) enriched in D1 cells and D2 cells are shown. Corresponding p-values and fold changes for DE performed for samples collected during the light and dark phase are shown for each transcript. Non-significant p-values for rhythmicity are also reported. Expression in D1 cells was used as the baseline for DE analysis.

Figure 6.. Model summarizing enriched rhythmic processes in MSNs across time of day and sex.

D1 and D2 cells taken from males and females have enriched rhythmic processes identified from transcripts peaking in the light and dark phase. Four clocks are shown, with gray shading indicating the dark phase (ZT12–0). Enriched processes are listed at the time of day in which transcripts peak during the light or dark phase for that cell type and sex.